Protocol for minimizing toxicity of combination dosages and imaging agent for verification

ABSTRACT

Advantage is taken of the enhanced permeability and retention effect (EPR effect) to shield normal tissue from exposure to combinations of chemotherapeutic agents. Imaging agents that exhibit the enhanced permeability and retention (EPR) effect in solid tumors are useful in mimicking the behavior of chemotherapeutic or other drugs for treatment of said tumor conjugated to carriers of similar size and shape to the carriers of said imaging agents.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. provisional application62/617,095 filed 12 Jan. 2018, U.S. provisional application 62/674,483filed 21 May 2018, U.S. provisional application 62/711,421 filed 27 Jul.2018, U.S. provisional application 62/716,788 filed 9 Aug. 2018, U.S.provisional application 62/716,796 filed 9 Aug. 2018, U.S. provisionalapplication 62/700,147 filed 18 Jul. 2018, and U.S. provisionalapplication 62/711,423 filed 27 Jul. 2018, the disclosures of which areherein incorporated by reference in their entirety.

TECHNICAL FIELD

The invention is in the field of combination treatments of solid tumorsand of diagnostic methods that assess pharmacokinetics of administeredentities, specifically with respect to the enhanced permeability andretention (EPR) effect exhibited when entities of nanometer dimensionsare administered to subjects with solid tumors. More specifically, theinvention relates to taking advantage of the EPR effect exhibited whenconjugates of nanometer dimensions are administered to subjects withsolid tumors.

BACKGROUND ART

Chemotherapeutic agents that are used to treat solid tumors are toxic tonormal tissue as well. Levels of such agents administered are limited bytheir maximum tolerated dose. When combinations of such agents are used,the toxicity of both agents is experienced by normal tissue whichfurther limits effective dosage levels. This problem has been addressedby designing protocols that avoid simultaneous administration of morethan one agent essentially on a trial-and-error basis which does notlead to optimal results. Another approach has been to utilizesynergistic combinations of two or more agents where their synergisticratio is maintained by controlling the pharmacokinetics using suitabledelivery vehicles, as set forth in U.S. Pat. Nos. 7,850,990 and9,271,931. Since the drugs are acting in synergy, lower dosage levelsare effective, thus also ameliorating the inherent toxicity of thedrugs.

Despite these approaches in the art, there remains a need for successfuldesign of protocols that will minimize the toxic effect of drugcombinations on normal tissue. The present invention solves this problemby taking advantage of the enhanced permeability and retention effect(EPR) of large molecules that can be used as carriers in order tocontrol exposure of normal tissue to the toxic drug and, by virtue ofthe present invention, assuring that the EPR effect is shown by theseconjugates.

As early as 1986, Maeda and coworkers demonstrated an EPR effect insolid tumors (Matsumura, Y., and Maeda, H., Cancer Res. (1986)46:6387-6392). Later work by this same group confirms this effect(Maeda, H., et al., J. Controlled Release (2001) 74:47-61; Maeda, H., etal., Eur. J Pharm. Biopharm. (2009) 71:409-419). Essentially, theseauthors showed that solid tumors growing beyond the size of a fewmillimeters in diameter depend on neovasculature that differs fromnormal vasculature in its architecture. While the cutoff pore size ofnormal vasculature is in the range of 2-6 nm, the neovasculature insolid tumors has a pore cutoff range of 100-700 nm (Dreher, M. R., etal., J. Natl. Cancer Inst. (2006) 98:335-344; Singh, Y., et al.,Molecular Pharmaceutics (2012) 9:144-155). The larger pores in the tumorneovasculature result in leakiness that allows macromolecules andnanoparticles to penetrate and extravasate into the tumor and thiscombined with poor lymphatic drainage results in the EPR effect whichresults in accumulation of macromolecules, conjugates or nanoparticlesthat is generally related to size and flexibility of the nanoparticle ormacromolecule and exposure (i.e., t_(1/2)). This has in particular beendemonstrated for liposomal delivery as noted, for example, by Allen, T.,et al., Science (2004) 303:1818-1822. Useful reviews of the literaturedescribing this effect include Danhier, F., et al., J. Control Rel.(2010) 148:135-146 and Eshun, F. K., et al., Gene Ther. (2010)17:922-929. With various size dextrans, it has been shown that there isan optimal size of 40- to 60 kDa and t_(in) (exposure time) thatprovides the most accumulation by the EPR effect (Dreher, M. R., et al.J Natl Cancer Inst (2006) Supra.)

In one aspect, the present invention relies on taking advantage of theEPR effect even for small molecules by providing conjugates tonanomolecular carriers, especially flexible carriers and by permittingdetermination of the pharmacokinetics associated with the EPR effect byproviding an imaging agent coupled to a carrier of similar dimensions tothose of a carrier used to deliver small molecules administered asconjugates to nanomolecular carriers, especially flexible carriers.

Jain, et al., have described features of the EPR effect relevant tonanomedicine design (Chauhan, V. P., and Jain, R. K., Nat. Mater. (2013)12:958-962; Chauhan, V. P., et al., Angew. Chem. Int. Ed. Engl. (2011)50:11417-11420; Chauhan, V. P., et al., Nat. Nanotechnol. (2012)7:383-388). Tumor vessel walls and tissue matrix exist as a series ofinter-connected pores with variable cross-sections. Cutoff sizes onlyindicate the largest particle that penetrates, and large particlesgenerally penetrate tumors heterogeneously and suboptimally comparedwith smaller particles. The vascular pore-size distribution within asingle tumor can vary by orders of magnitude, with most of the poresactually being much smaller than the pore cutoff size. Thus, theeffective vascular permeability of small particles does not necessarilycorrelate with cutoff size; smaller particles penetrate tumors morerapidly and uniformly than larger particles and smaller particlescarrying drugs should be more generally effective against solid tumorsthan larger particles.

The shape of the nanoparticles also modifies the EPR effect (Chauhan, V.P., (2011) supra). Non-spherical nanoparticles can penetrate tumors morerapidly and accumulate at higher levels than size-matched spheres,because of enhanced penetration through the pores is related to theshortest dimension of the particle. The advantage of non-sphericalparticles holds for smaller vessel-pore-sizes but is lost with respectto large pore sizes.

Many or most studies of nanoparticles for EPR drug delivery and imagingutilize larger 100 nm liposomes/particles containing appropriate drugsor isotopes. As described above, regardless of cut-off pore size theselarger nanoparticles are likely not the optimal size for accumulation inmany tumors since most will contain neovasculature with heterogeneouspore sizes; thus the present invention is focused on carriers withhydrodynamic radii of less than 50 nm.

The present invention, in some embodiments, employs linking technologiesthat are particularly favorable for preparation of conjugates designedto take advantage of the EPR effect. In particular, linkages thatrelease a small molecule chemotherapeutic agent (drug) by betaelimination have been disclosed. See, for example, U.S. Pat. Nos.8,680,315; 9,387,254; 8,754,190; 8,946,405; and 8,703,907, and WO2015/051307, all incorporated herein by reference. Such linkers permittuning of the time of release of the coupled drug by adjusting theacidity of a carbon-hydrogen bond positioned beta to a suitable leavinggroup.

It has also been possible to study this effect by using detectablemarkers coupled to nanoparticles. Wilks, M. Q., et al. (Bioconjug. Chem.(2015) 26:1061-1069) reported a 30 kDa PEG-DFB-⁸⁹Zr conjugate (alsocontaining fluorescent Cy5.5). In the mouse, it showed an eliminationt_(1/2) of 13.5 hr and high retention (˜4 to 5% ID/g) in an implantedHT-29 tumor at 48 hr post injection. The kinetics of tumor accumulation,clearance or capacity were not determined. Because these nanoparticlesare only about 10 nm and are flexible, their biological distributiondoes not show a strong EPR effect in tumor tissue. However, this studyshows that labeled conjugates can be thus used to elucidate theseparameters.

Another technology useful in the method of the invention is positronemission tomography (PET) which offers some advantages over the use offluorescent label for such studies. Current knowledge on the EPR effectin human tumors is largely based on studies of low-resolution singlephoton imaging techniques of radiolabeled liposomes c.f. (Harrington, K.J., et al., Clin. Cancer Res. (2001) 7:243-254; Khalifa, A. et al.,Nucl. Med. Commun. (1997) 18:17-23), which could visualize tumors butcould not quantitate the EPR effect. The high detectionsensitivity/quantitation and spatial resolution of PET make thistechnology superior for quantitative studies of nanoparticlebiodistribution. For example, Lee H, et al., Clin Cancer Res23(15):4190-4202, showed that 64Cu-labeled HER2-targeted liposomaldoxorubicin—about/over 100 nm diameter—accumulated in human tumors andcould be quantified by PET. The range of intra- and inter-patient tumordrug concentrations measured was proposed to result in variableantitumor activity of these liposomes that included both a therapeuticand diagnostic (PET labeled) moieties, designated herein theranosticnanoparticles (TNP). Tumor deposition was stratified and uptake levelswere retrospectively associated with treatment outcomes: high uptaketumors were susceptible to the effect of the TNPs (75% partialremission/stable disease) whereas low-uptake tumors (43% stable disease)were not. Brain metastases were also imaged, suggesting theirvasculature had increased pore sizes that could make such metastasissusceptible to TNPs. These results indicate that a NP imaging approachmay be applicable as a predictive strategy for personalizingnanomedicines, whereby a diagnostic procedure is performed, and thenonly patients with susceptible tumors are treated with the TNPs. Insummary, these data suggest that it may be possible to use pretreatmentimaging of NP deposition in tumors to identify patients most likely tobenefit from treatment with closely related TNPs.

Using these tools available in the art, protocols are constructed thatameliorate the toxic effect of combination therapy on normal tissue.

DISCLOSURE OF THE INVENTION

One goal of the invention is to confine the cytotoxic effect of drugsadministered in combination to tumor tissue while sparing normal tissueto the extent possible. In one approach, this can be done by adjustingthe dosage administration protocol so that while a firstchemotherapeutic agent is sequestered in a solid tumor and no longeravailable in the system to exert an effect on normal tissue a secondtherapeutic agent is administered so that effectively only the toxiceffects of the second drug, without supplementation by the first, areexerted in the system while the combined effects are exerted in thetumor. In a second approach, both agents are sequestered as conjugatesin the solid tumor so that higher concentrations of both agents areexperienced by tumor cells than are experienced by normal tissue and theagents are cleared from normal tissue while remaining in the tumor.

Thus, in one aspect, the invention is directed to a method to amelioratethe toxicity to normal tissue in a subject resulting from administeringto said subject a first and second chemotherapeutic agent in a protocolfor combination therapy against a solid tumor employing said first andsecond agent, which method comprises:

administering the first agent as an agent-releasing conjugate to acarrier, wherein the carrier is a nanoparticle or macromolecule eachwith a hydrodynamic radius of 5-50 nm (i.e., a diameter of 10-100 nm)which conjugate exhibits enhanced permeability and retention (EPR) insolid tumors so as to concentrate said conjugate in the tumor andwherein the rate of release from the tumor of the conjugate and firstagent released from the conjugate is substantially slower than the rateof clearance of the conjugate and released agent from the systemiccirculation of the subject;

allowing a time period for clearance of the conjugate and released agentfrom the systemic circulation of the subject; and

after said time period, administering said second agent to the subject.

In some embodiments, an additional agent that has a non-overlappingtoxicity with the second agent may also be administered.

In a second aspect, the invention is directed to a method to minimizethe toxic effects on normal tissue of a subject of a first and secondchemotherapeutic agent used in combination to treat a solid tumor insaid subject which method comprises administering both said first andsecond agents as releasable conjugates to carriers, wherein the carriersare nanoparticles or macromolecules each with a hydrodynamic radius of5-50 nm (10-100 nm diameter) wherein said conjugates exhibit enhancedpermeability and retention (EPR) and effect concentration of both saidconjugates in said tumor.

In some embodiments of the simultaneous administration, only the firstagent is conjugated and the second agent is in unconjugated form.

In some instances, a third similarly conjugated or unconjugatedtherapeutic agent may be employed as well.

In connection with the foregoing methods, when the second or third agentis conjugated the carriers mimic those of the first agent. In any case,labeled non-releasable conjugates comprising carriers with the samecharacteristics as those used in conjugating the drugs can be used tomonitor the uptake of the conjugates by the solid tumor. Administeringsuch conjugate where the carrier is non-releasably linked to the labelpermits verification (or not) that the corresponding conjugates of drugswill exhibit an EPR effect. The labels used in such monitoring arepreferentially those detectable by positron emission tomography (PET).

Thus, the present invention also offers a method to mimic thepharmacokinetics of a conjugate of a drug with respect to its behaviorin the context of an EPR effect in solid tumors. By providing a suitableimaging agent with a carrier similar in size and shape to a carrierconjugated to a drug, the pharmacokinetics of the drug can be predictedby monitoring the pharmacokinetics of the imaging agent. Such diagnosticagents are also useful in the determining the suitability of treatingpatients with conjugates of therapeutic agents.

Thus, in one aspect, the invention is directed to an imaging agent ofthe formula (1)

wherein PEG represents a polyethylene glycol comprising a plurality of2-6 arms of 40-60 kD;

chelator represents a desferrioxamine or a plur-hydroxypyridinonemultidentate;

I is a radioisotope suitable for positron emission tomography (PET);

is a covalent connector;

˜ indicates sequestration of I in the chelator; and

n is an integer of 1 up to the number of arms of said PEG.

The invention also includes hybrid conjugates of formula (2)

wherein PEG represents a polyethylene glycol comprising a plurality of2-6 arms of 40-60 kD;chelator represents a desferrioxamine or a plur-hydroxypyridinonemultidentate;I is a radioisotope suitable for positron emission tomography (PET);

is a covalent connector;˜ indicates sequestration of I in the chelator;L is a linker;D is a therapeutic agent;n is an integer of 1 up to the number of arms of said PEG minus x, andx is an integer of up to the number of arms of said PEG minus n.

The use of a multi-armed PEG is advantageous in that the resultingnanoparticle is less flexible, and thus retained more preferentially intumors. The imaging agent will optimally have a diameter ofapproximately 20 nm (a hydrodynamic radius of approximately 10 nm). Thediameter can be in the range of 10-100 nm, or 10-50 nm or 10-25 nm,corresponding to hydrodynamic radii of 5-50, 5-25 or 5-12.5 nm.

In another aspect, the invention is directed to a method to monitoraccumulation of the imaging agent in a tumor which method comprisesadministering said imaging agent and detecting the location of saidimaging agent by PET.

In still another aspect, the invention is directed to a method to assessthe pharmacokinetics of a drug conjugate and its accumulation in tumorwhich method comprises matching the size of a conjugate of said drug tothe size of the imaging agent, administering said imaging agent andmonitoring the accumulation of said agent in the tumor by PET asdiagnostic of the behavior of the drug conjugate.

Thus, the invention further includes method to assess suitability oftreating a patient with a conjugated drug based on the diagnostic agent.The dimensions of the diagnostic agent are matched to those of a drugconjugate intended for patient treatment. More broadly the diagnosticagent can simply identify patients that can be treated taking advantageof the EPR effect.

The invention also includes kits that include the imaging agent of theinvention and a conjugate of a drug of similar size and shape as theimaging agent.

In another aspect, the invention is directed to a method to identify asubject that will likely benefit from treatment with a drug modified toexhibit the EPR effect, which comprises administering the imaging agentof the invention to a candidate subject and monitoring the distributionof the imaging agent in the subject, whereby a subject that accumulatessaid imaging agent in an undesirable tissue mass is identified as asubject that will benefit from such treatment. See, for example, Lee,H., et al., Clin. Canc. Res., (2017) 23:4190-4202 (supra).

In connection with the protocols for treatment, the imaging agents ofthe invention having carriers with the same characteristics as thoseused in conjugating the drugs are used to monitor the uptake of theconjugates by the solid tumor. This permits verification (or not) thatthe corresponding conjugates of drugs will exhibit an EPR effect.

In a further aspect the invention includes a method to identify asubject having a tumor that will respond to treatment with an inhibitorof DNA repair which method comprises determining the presence or absenceof a mutation in a gene that encodes a protein that participates ineffecting DNA repair, wherein the presence of said mutation in thesubject identifies the subject as having such a tumor.

In still another aspect the invention is directed to a hybrid conjugatefor treatment and imaging of solid tumors which conjugate comprises aflexible carrier wherein the carrier is a nanoparticle or macromoleculeeach with a hydrodynamic radius of 5-50 nm which conjugate exhibitsenhanced permeability and retention (EPR) in solid tumors so as toconcentrate said conjugate in the tumor and wherein said carrier isreleaseably coupled to a therapeutic agent and also to an imaging agent,and to a method to correlate imaging and treatment of a solid tumorusing said hybrid conjugate. An exemplary generic structure of suchhybrids for any drug such hybrids designated as “theranostics” is shownin FIG. 12.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the concentration of coupled SN-38 in the formof a conjugate to a four-armed 40 kD PEG (PLX038) in the plasma as afunction of time. Similar results for the released SN-38 and thedetoxified form of the drug, i.e., the glucuronide (SN-38G) are shown inthe same figure. The rates are similar showing half-lives of 50 hours inthe rat.

FIG. 2 shows the effect of various concentrations of PLX038 administeredto the HT29 xenograft-bearing rat as compared to irinotecan.

FIGS. 3A and 3B show the concentrations of PLX038 in free SN-38 atvarious dosages in the tumor as compared to plasma.

FIG. 4 is a diagram showing a hypothetical dosing schedule in humans ofa combination of PLX038 and a second drug (e.g., a poly ADP ribosepolymerase (PARP) inhibitor) administered systemically. PLX038 isadministered on day 1; the conjugate accumulates in the tumor andreleases the free drug (dotted line) in the vicinity of the tumor andboth conjugate and free drug are cleared from the system (solid line).After 2 half-lives of systemic clearance, in this case 10 days, systemicPLX038 is reduced to 25% of its original concentration, and theconcentration lies below its minimal effective (and toxic) level. Atthis time the second drug is administered on an effective schedule.

FIG. 5 shows C vs. t plots of SN-38 released from PLX038 in the rat andfrom PLX038A in mouse. The curve for SN-38 released from PLX038 at 3.2nmol (200 mg)/kg in the rat was modeled using previously determinedpharmacokinetic parameters (Santi, D. V., et al., Proc. Natl. Acad. Sci.USA (2012) 109:6211-6216).

FIGS. 6A-6E are maximum intensity projections (MIP) at 72h and 120h ofPEG40 kDa-DFB-⁸⁹Zr in mice bearing HT-29 xenografts (A) on both flanksoverlaid on a CT scan; ex vivo biodistribution study of PEG40kDa-DFB-⁸⁹Zr in mice bearing HT-29 xenografts (B) and tumor to bloodratios (C) vs time in mice bearing HT-29 tumors; 72h MIP image (D) ofPEG-(SN-38)3-DFB-⁸⁹Zr in single flank tumor bearing mice andbiodistribution of PEG-(SN-38)3-DFB-⁸⁹Zr (black) vs PEG-DFB-⁸⁹Zr (grey)at 72h (E).

FIGS. 7A-7C show the biodistribution of PEG_(40kDa)-(DFB)-⁸⁹Zr₄ in micebearing tumors.

FIG. 8 shows the biodistribution of various ⁸⁹Zr conjugates in HT-29xenografts.

FIG. 9 shows the biodistribution of various ⁸⁹Zr conjugates in MX-Ixenografts.

FIG. 10 shows the effectiveness of PEG-SN38 in tumor treatment.

FIGS. 11A-11C show synergy of an SN38 conjugate and separatelyadministered talazoparib.

FIG. 12 shows a generic hybrid drug/label conjugate theranostic.

MODES OF CARRYING OUT THE INVENTION

Essentially, there are two approaches to the design of protocols thatminimize the toxic effects of combination therapies. The first approachis to ensure that a first therapeutic agent or drug is captured in asolid tumor to be treated by coupling the drug to a carrier such thatthe EPR effect results in substantially retaining the conjugate andreleased drug in the solid tumor, while the administered conjugate andreleased drug not captured in the tumor are more rapidly cleared fromthe systemic circulation, wherein the carrier is a nanoparticle ormacromolecule each with a hydrodynamic radius of 5-50 nm preferablyabout 10 nm (diameter of 10-100 nm preferably about 20 nm). Thus asubstantial portion of the administered conjugate is retained in thetumor, as well as is the drug that has been released from the conjugatewhile the conjugate resides in the tumor. As the clearance rate from thesystemic circulation is much greater than the clearance rate of theconjugate and released drug from the tumor, an effective amount of drugboth in conjugated and free form remain to exert a cytotoxic effect ontumor cells while their concentration in the systemic circulation hasdiminished to a desired level. After two half-lives in the systemiccirculation, for example, the level of the conjugate and free drug incirculation and in contact with normal tissue is reduced to 25% of theinitial concentration, and this may be sufficiently low to amelioratetoxicity. Since the conjugate remains in the tumor to release the agent,the agent is able to exert its cytotoxic effect on the tumor althoughits concentration in the systemic domain is quite low, and exposure ofnormal tissue to the drug is therefore also quite low.

At this point, a second drug is administered systemically and thus thenormal tissue is exposed only to the toxic effect of the second drugwhile the first drug remains out of reach in the tumor. This minimizesthe toxic effect of the combination on normal tissue while retaining thecombined toxicities in the tumor. The second drug may be administeredeither in free form or it, too, may be administered as a conjugate witha similar carrier or in any other suitable form, including inclusion indelivery vehicles such as liposomes, nanoparticles, micelles, and thelike.

In addition, a third drug that has non-overlapping toxicity with thesecond drug may be coadministered simultaneously or sequentially withsaid second drug.

Alternatively, both the first and second drug may be administered in theform of conjugates that are retained in the tumor by virtue of EPReither at the same time or at disparate times. By virtue of thisretention, the major concentration of each drug occurs in the tumorrather than being in contact with normal tissue. Thus, the higher dosagelevels of these drugs is experienced mainly in the tumor, and theadministered conjugates along with released drug are rapidly clearedfrom the systemic circulation.

In some instances, still an additional conjugated form of an agent maybe coadministered.

The carriers used in the method of the invention to administer at leastthe first agent in the first above-cited method and to release both thefirst and second agents in the second-noted method are carriers that areflexible in nature and have hydrodynamic radii of about 10 nm. Suitablemacromolecule carriers include polyethylene glycols (PEG) which may belinear or multi-armed and have molecular weights of 10-50 kD.Preferably, the carriers are multi-armed PEG with molecular weights ofat least 20 kD. These characteristics of the carriers assure thatmaximum advantage can be taken of the EPR effect. Nanoparticulatecarriers are also included.

Particularly useful to provide a releasable form of a conjugate of thechemotherapeutic agents to nanomolecular carriers are linkers thatrelease the agent by beta elimination reactions such as those describedin detail in the above cited U.S. Pat. Nos. 8,680,315; 9,387,254;8,754,190; 8,946,405; and 8,703,907 all incorporated herein by referencefor their disclosures of not only the structure of useful linkers thatrelease the agent by beta elimination, but also with respect to theirdisclosure of nanomolecular carriers useful in the present invention aswell.

Other linkers include those cleavable by hydrolysis of esters,carbonates, or carbamates, by proteolysis of amides or by reduction ofaromatic nitro groups by nitroreductase.

The subjects of the methods of the invention are typically humansubjects, but the invention methods are also applicable in veterinarycontexts including livestock and companion animals. The methods are alsosuitable for animal models useful in the laboratory such as rats, mice,rabbits or other model systems preparatory to designing protocols forhuman use.

With respect to the drugs useable in the combination therapy, a widevariety of chemotherapeutic agents is known and any combination of thesemay be selected as the first and second drug. Agents that act additivelyor synergistically are preferred, for example combination of drugswherein each inhibits DNA repair.

Drugs that cause DNA damage, such as Topo 1 inhibitors, are particularlyuseful in treating tumors whose genome contains a mutation in a genethat normally aids in DNA repair. Among others, these genes includeBRCA1, BRCA2, ATM which encodes ataxia telangiectasia mutated (ATM)kinase and ATR which encodes Rad-3 related (ATR) kinase. The inventionincludes identifying tumors that will show enhanced sensitivity totreatment with a Topo 1 inhibitor where the tumor-bearing subject'sgenome has at least one gene that has a mutation in BRCA1, BRCA2, ATM orATR or other genes where mutation prevents or depresses the ability ofthe gene to enhance DNA repair. The response may be further enhanced byinhibiting a second enzyme involved in DNA repair, such as a PARPinhibitor, which then causes a synthetic lethality that is amplifiedbecause of the high level of DNA breaks caused by the Topo inhibitor.Thus, in using passively targeted PEG_SN38, it is useful to know thegenetic status of the tumor, and to have an assortment choice ofinhibitors of the DNA damage response system.

Examples of Agents Include:

“Signal transduction inhibitors” which interfere with or prevent signalsthat cause cancer cells to grow or divide;

“Cytotoxic agents”;

“Cell cycle inhibitors” or “cell cycle control inhibitors” theseinterfere with the progress of a cell through its normal cell cycle, thelife span of a cell, from the mitosis that gives it origin to the eventsfollowing mitosis that divides it into daughter cells;

“Checkpoint inhibitors” these interfere with the normal function of cellcycle checkpoints, e.g., the S/G2 checkpoint, G2/M checkpoint and G1/Scheckpoint;

“Topoisomerase Inhibitors”, such as camptothecins, which interfere withtopoisomerase I or II activity, enzymes necessary for DNA replicationand transcription;

“Receptor tyrosine kinase inhibitors” these interfere with the activityof growth factor receptors that possess tyrosine kinase activity;

“Apoptosis inducing agents” these promote programmed cell death;

“Antimetabolites,” such as gemcitabine or hydroxyurea, which closelyresemble an essential metabolite and therefore interfere withphysiological reactions involving it;

“Telomerase inhibitors” these interfere with the activity of atelomerase, an enzyme that extends telomere length and extends thelifetime of the cell and its replicative capacity;

“Cyclin-dependent kinase inhibitors” these interfere withcyclin-dependent kinases that control the major steps between differentphases of the cell cycle through phosphorylation of cell proteins suchas histones, cytoskeletal proteins, transcription factors, tumorsuppresser genes and the like;

“DNA damaging agents”;

“DNA repair inhibitors”;

“Anti-angiogenic agents”, which interfere with the generation of newblood vessels or growth of existing blood vessels that occurs duringtumor growth; and

“Mitochondrial poisons” which directly or indirectly disruptmitochondrial respiratory chain function.

Many combinations of these for treatment of tumors are the clinicallyapproved.

Preferred agents that may be used in combination include DNA damagingagents such as carboplatin, cisplatin, cyclophosphamide, doxorubicin,daunorubicin, epirubicin, mitomycin C, mitoxantrone; DNA repairinhibitors including 5-fluorouracil (5-FU) or FUDR, gemcitabine andmethotrexate; topoisomerase I inhibitors such as camptothecin,irinotecan and topotecan; S/G2 or G2/M checkpoint inhibitors such asbleomycin, docetaxel, doxorubicin, etoposide, paclitaxel, vinblastine,vincristine, vindesine and vinorelbine; G1/early S checkpointinhibitors; G2/M checkpoint inhibitors; receptor tyrosine kinaseinhibitors such as genistein, trastuzumab, ZD1839; cytotoxic agents;apoptosis-inducing agents and cell cycle control inhibitors.

Exemplary combinations are DNA damaging agents in combination with DNArepair inhibitors, DNA damaging agents in combination with topoisomeraseI or topoisomerase II inhibitors, topoisomerase I inhibitors incombination with S/G2 or G2/M checkpoint inhibitors, G1/S checkpointinhibitors or CDK inhibitors in combination with G2/M checkpointinhibitors, receptor tyrosine kinase inhibitors in combination withcytotoxic agents, apoptosis-inducing agents in combination withcytotoxic agents, apoptosis-inducing agents in combination withcell-cycle control inhibitors, G1/S or G2/M checkpoint inhibitors incombination with cytotoxic agents, topoisomerase I or II inhibitors incombination with DNA repair inhibitors, topoisomerase I or II inhibitorsor telomerase inhibitors in combination with cell cycle controlinhibitors, topoisomerase I inhibitors in combination with topoisomeraseII inhibitors, and two cytotoxic agents in combination.

Exemplary specific agents include cisplatin (or carboplatin) and 5-FU(or FUDR), cisplatin (or carboplatin) and irinotecan, irinotecan and5-FU (or FUDR), vinorelbine and cisplatin (or carboplatin), methotrexateand 5-FU (or FUDR), idarubicin and AraC, cisplatin (or carboplatin) andtaxol, cisplatin (or carboplatin) and etoposide, cisplatin (orcarboplatin) and topotecan, cisplatin (or carboplatin) and daunorubicin,cisplatin (or carboplatin) and doxorubicin, cisplatin (or carboplatin)and gemcitabine, oxaliplatin and 5-FU (or FUDR), gemcitabine and 5-FU(or FUDR), adriamycin and vinorelbine, taxol and doxorubicin,flavopiridol and doxorubicin, UCN-01 and doxorubicin, bleomycin andtrichlorperazine, vinorelbine and edelfosine, vinorelbine andsphingosine (and sphingosine analogues), vinorelbine andphosphatidylserine, vinorelbine and camptothecin, cisplatin (orcarboplatin) and sphingosine (and sphingosine analogues), sphingosine(and sphingosine analogues) and daunorubicin and sphingosine (andsphingosine analogues) and doxorubicin.

In one embodiment, for a first drug is a releasable conjugate of theinvention of SN-38, a topoisomerase inhibitor, exemplary second drugsinclude PARP inhibitors, mTOR inhibitors, trabectedin, cis-platinum,oxaliplatin, fluorouracil, temozolomide and vincristine all of whichhave been reported to be synergistic with SN-38.

Certain tumors are especially susceptible to treatment with PARPinhibitors and in these tumors, PARP inhibitors are favored as thecombination drug. These are tumors wherein a mutation in a gene thatnormally is helpful in providing a protein that aids in DNA repair takesaway this property of the gene. Such tumors are also responsive totopoisomerase inhibitors, such as SN38, since inhibition oftopoisomerase causes excess DNA damage that requires DNA repair that isdeficient in these tumors. These genes include BRCA1, BRCA2, ATM whichencodes ataxia telangiectasia mutated (ATM) kinase and ATR which encodesRad-3 related (ATR) kinase, among others. The invention includesidentifying tumors that have mutations in BRCA1, BRCA2, ATM or ATR orother genes where mutations prevent or depress the ability of the geneto enhance DNA repair and combining treatment with the invention SN38conjugates with follow up treatment with for example PARP inhibitors, orother inhibitors of DNA repair. Because the drug accumulates and remainsin the tumor after it is eliminated from the rest of the system, thetoxicity of the SN38 is confined to the tumor and the system as a wholehas only to deal with toxicity of the PARP inhibitor.

Some of the above listed drugs to be administered as second drugs may beadministered in combination either sequentially or simultaneouslyprovided their toxicities do not overlap.

Imaging

Since the invention methods rely on the ability of the conjugatesadministered for the first agent in the first approach above and boththe first and second agents in the second approach being subject to theEPR effect, it is important to confirm that this is in fact the casesince tumors are heterogeneous and the particular carrier selected mustbe compatible with the pore structure of the vasculature in the solidtumor that resides in the subject in the sense that the EPR effect ispresent. Therefore, in some embodiments of the invention method, this isconfirmed by administration either at the same time or separately of aconjugate of a label that is coupled non-releasably to the same carrieror a carrier with the same characteristics as that linked to thedrug(s). While any detectable label, e.g., fluorescent label, can beused, it is most convenient to employ an isotope that is detectable bypositron emission tomography (PET) scanning. The non-releasableconjugate of the isotope is then monitored to detect whetherpreferential uptake and retention by the tumor is exhibited. If so, themethod of the invention is employed. If the tumor fails to exhibit theEPR effect with the labeled non-releasable conjugate, the method of theinvention is contraindicated. The isotopes thus detectable are wellknown in the art as are means for coupling such isotopes tomacromolecular carriers.

For imaging, a similar conjugate is used. As noted above, it isadvantageous to design the imaging agent of the invention such that thediameter is approximately are 20 nanometers and to avoid excessiveflexibility. This can be accomplished by using the multi-armed PEGpolymers in the range of 40-60 kD. Although the number of armsassociated with this polymer may range from 1-6, multi-armed PEGs of 3-5arms, more preferably 4 arms are focused on herein.

The value of n in formula (1) can vary from 1 to the number of armsassociated with the polymer and it should be understood that in thecompositions of the invention the value of n may not be the same for allof the individual imaging moieties in the composition. Thus, forexample, for a 4 armed PEG where n is 4, or in single chain PEG where nis 1, most of the individual “molecules” in a given composition willcontain 4 or 1 as values of n respectively. However, for example for 4armed PEG, for n=3 or n=2, represents an average and some of theindividual entities may comprise 4, some comprise 3, some comprise 2 andsome comprise 1 instances of n value

Further as to the structure of the imaging agent of Formula (1) notedabove, the chelator represents a desferrioxamine or a multidentatechelator comprised of a multiplicity of hydroxypyridinones, abbreviatedherein “plur-hydroxypyridinone multidentates.” A variety of suchchelators are well known in the art and are described in detail, forexample, in Ma, M. T. et al., Dalton Trans (2015) 44:4884-4900 and byDeri, M. A., J Med Chem (2014) 57:4849-4860. The description of theseligands in these documents is specifically incorporated herein byreference.

The covalent connector on Formula (1) may be a direct bond to thechelator or there may be intermediate linkers such as dipeptides orbifunctional linkers comprising 1-20 linking atoms. Radioisotopes (I)useful in PET in the context of the present invention are known in theart, and particularly a subset preferred among those set forth in Table3 of Smith, S. V. et al., “Production and Selection of Metal PETRadioisotopes for Molecular Imaging,” in Radioisotopes Applications inBio-Medical Science, Nirmal Singh, ed., Chapter 10, InTech (Rijeka,Croatia), 2011, are those with suitable half-lives such as ⁸⁹Zr, ⁹⁴Tc,¹⁰¹In, ⁸¹Rb, ⁶⁶Ga, ⁶⁴Cu, ⁶²Zn, ⁶¹Cu or ⁵²Fe.

To use the imaging agents of the invention as surrogates for delivery ofactive agents, i.e. drugs, the imaging agents contain carriers with thesame characteristics as those carriers used in conjugating the drugs.These are then used to monitor the uptake of the conjugates by the solidtumor. This permits verification (or not) that the correspondingconjugates of drugs will exhibit an EPR effect.

An alternative to using separate therapeutic and imaging conjugatesemploys a hybrid conjugate of formula (2) for treatment and imaging ofsolid tumors which conjugate comprises a flexible carrier wherein thecarrier is a nanoparticle or macromolecule each with a hydrodynamicradius of 5-50 nm which conjugate exhibits enhanced permeability andretention (EPR) in solid tumors so as to concentrate said conjugate inthe tumor and wherein said carrier is releasably coupled to atherapeutic agent and also coupled to an imaging agent. Thus, in formula(2) as in formula (1),

in some embodiments I is ⁸⁹Zr, ⁹⁴Tc, ¹⁰¹In, ⁸¹Rb, ⁶⁶Ga, ⁶⁴Cu, ⁶²Zn, ⁶¹Cuor ⁵²Fe, and/or the PEG is a four armed polyethylene glycol ofapproximately 40 kD, and n is 1-4, and/or the chelator isdesferrioxamine-B, and/or

is a direct bond linkage.

As shown, at least one of the arms of the PEG is occupied by the imagingagent and at least one is occupied by the therapeutic agent. Variouscombinations up to the total number of arms of the PEG polymer arecontemplated. The therapeutic agent may be SN38 or other topoisomeraseinhibitor or any other suitable agent for tumor treatment that isbenefited by accumulation in the tumor, such as a PARP or kinaseinhibitor.

The imaging agents of the invention are also useful to identify subjectsthat harbor tumors or other tissue masses that are susceptible totreatment with therapeutic agents that exhibit the EPR effect. Thus, theimaging agent may be administered to a subject and monitored todetermine whether the tumor, for example, will, in fact, preferentiallytake up and retain entities of similar size.

In this application, “a”, “an”, and the like are intended to mean one ormore than one unless it is clear from the context that some othermeaning is intended. In addition, the terms “chemotherapeutic agent”,“agent”, and “drug” are used interchangeably. Where specific numericalcharacteristics are set forth, the number cited will typically haveerror bars of plus-or-minus 10%, preferably plus-or-minus 5% and morepreferably plus-or-minus 1%. Thus, a range of 10-50 nm could include arange of 9-55 nm. “Hydrodynamic radius” means the apparent Stokes radiusthe radius of a hard sphere that diffuses through solution at the samerate as the molecule in question as measured, for example, by gelpermeation chromatography.

The subjects of the invention are typically human, but also includenon-human animals such as laboratory models and veterinary subjects.

All documents cited herein are hereby incorporated herein by reference.

The following examples are offered to illustrate but not to limit theinvention.

Example 1 Administration of Conjugated SN-38

SN-38 is the topoisomerase I inhibitor that is the active drug releasedfrom the prodrug, irinotecan. Conjugates of SN-38 are described in WO2015/051307. Two different conjugates of SN-38 were prepared: PLX038 andPLX038A. These conjugates couple the drug releasably to a four-armed PEGof 40 kD through a linker that effects release by β-elimination. Thestructure of PLX038 and PLX038A is shown below wherein “Mod” is CN inPLX038, and methyl sulfonyl in PLX038A.

Six rats bearing HT29 tumor xenografts were injected with 200 mg/kg ofPLX038 and the concentration in plasma and tumor of the conjugate andreleased drug as well as its glucuronide (SN-38G) were followed by HPLCwith fluorescence monitoring As shown in FIG. 1, the half-life of PLX038in the systemic circulation is about 50 hours. The conjugate and thefree drug as well as SN-38G show similar half-lives.

As shown in FIG. 2, the efficacy of a non-toxic dose of 20 nmol/kg ofSN-38 in the form of PLX038 exceeds that of a toxic gastrointestinaldose of irinotecan control.

This is explained by the results shown in FIGS. 3A and 3B which aregraphs of the levels of the conjugate PLX038 and of SN-38 that has beenreleased from the conjugate in the tumor at various dosage levels. Asseen in FIG. 3A, at an administered dose of 200 mg/kg the level ofPLX038 in the tumor (a left bar) is roughly 8 nmol/g while theconcentration in the plasma (shown as the right bar) is barelydetectable. Similarly, in FIG. 3B with respect to the released SN-38, atthe same dosage, the left bar shows the concentration in the tumor asabout 80 pm/g while, again, the right bar shows that in the circulationit is barely detectable. Indeed, as shown, at the lower dosages, theconjugate and free drug are not detected in the plasma, while the tumorshows significant concentrations of these moieties.

Example 2 Suggested Human Protocol

A dosing schedule in humans for a combination of PLX038 and a seconddrug (e.g., a PARP inhibitor) administered systemically is proposedwherein PLX038 is administered on day 1 whereby the conjugateaccumulates in the tumor and releases the free drug. The conjugate andthe free drug are concomitantly cleared from the system. After twohalf-lives of systemic clearance or 10 days, systemic PLX038 is reducedto 25% of its original concentration, which lies below its minimaleffective and toxic levels. At this time the second drug, which issynergistic with SN-38 is administered orally for 20 days.

As shown in FIG. 4, the EPR effect concentrates PLX038 in the tumor(dotted line), while the systemic PLX038 (solid line) is sufficientlylow that any toxic effect is only to a second drug, which isadministered as shown at 10 days. At that time, the concentration of theconjugate in the tumor is still above the minimum effective level butbelow the toxic level.

Example 3 Design of a Mouse Model

Because most xenograft tumor models use mice as hosts, it is desirableto adapt the protocols of the present invention to testing in mice.Adaptation is needed because the half-life of the PLX038 conjugate inthe mouse is only about 24 hours, whereas in the rat it is about 48hours and in humans about 6 hours. Because the more rapid elimination ofPLX038 in mice occurs before substantial amounts of the SN-38 arereleased, a different conjugate of SN-38, PLX038A that has a highercleavage rate, is used in murine experiments.

Linker cleavage is species independent. While 32% of PLX038 is convertedto SN-38 over one half-life of the conjugate in humans, only 12% isconverted in the rat and 6% in the mouse. For PLX038A, the cleavagehalf-life is 70 hours and 26% conversion to SN-38 over one half-life ofthe conjugate in the mouse occurs. This conjugate also can beadministered intraperitoneally (IP) in mice with 100% bioavailability.

However, in mice PLX038A still has a short t_(1/2) of renal eliminationso a single dose may not effect high tumor accumulation and longerexposure may be necessary to achieve this. A multi-dose schedule forPLX038A in the mouse that simulates a single effective dose of theconjugate that gives high tumor accumulation in the rat is thereforeused.

For comparison, in the rat xenograft model for colon cancer (HT-29), asingle 200 mg/kg of PLX038 produced 61% inhibition of tumor growth withno gastrointestinal (GI) toxicity while irinotecan control that showednear-equal tumor inhibition showed significant GI toxicity. There washigh accumulation of PLX038 and SN-38 in tumors 14 days post-dosing whenthe serum levels were undetectable. A dosing schedule for PLX038A in themouse that would simulate the pharmacokinetics (PK) of PLX038 in the ratis shown in FIG. 5. Three daily decreasing doses of 152, 60 and 54mmol/kg effectively simulate the rat PK profile of released SN-38 fromPLX038. The “effective” half-life of SN-38 in the schedule is over 2days, whereas SN-38 from irinotecan in the mouse is 2 hours. The datasupporting FIG. 5 are shown in Table 1.

TABLE 1 mouse dose conj dose, SN-38 SN-38 dose, schedule mg dose, mgnmol dose 1 1.7 0.060 152 dose 2 0.9 0.032 80 dose 3 0.6 0.021 54 total3.2 0.1 285.4 conj SN-38 SN-38 time dose, dose, dose, AUC, over mg/kgmg/kg μmol μM-h 8 nM rat dose 200 7 3.2 11 ~7 days mouse total 128 4.50.285 11 ~5 dose days

Example 4 Murine Testing

The ability of HT29 xenografts to accumulate conjugate using the EPReffect is tested by injecting mice with one dose IP of 50 nmol of 40 kDfour-armed PEG fluorescein per 100 g (15 nmol/mouse) to obtain about 10μM in serum. Blood and tumor are sampled at various times (at 6, 24, 48and 96 hours) and the level of fluorescein measured. (The tumor isexcised and digested with sodium hydroxide for measurement.)

PLX038A is tested for tumor growth inhibition in a nude mouse HT29 tumorxenograft using the three-dose schedule developed in Example 3.

The nude mouse model with HT29 xenograft is treated with the three-doseschedule of PLX038A developed in Example 3 and at 14 days the mice weretreated daily with oral administration of a PARP inhibitor.

A conjugate of PARP inhibitor analogous to PLX038A is administered dailyto nude mice bearing HT29 tumors and tested vs. daily administration offree inhibitor.

Combinations of conjugates PLX038A and the relevant conjugate of PARPinhibitor are also tested concomitantly in this model.

Example 5 Synthesis of PEG_(40kDa)-PET isotopes

Synthesis of PEG-Desferrioxamine Conjugates

4-Branched PEG_(40kDa) Coupled to DFB:

A solution of 4-branched 40-kDa PEG-amine (GL4-400PA, NOF; 150 mg, 3.75umol) and p-isothiocyanatobenzyl-desferrioxamine B (Macrocyclics; 4 mg,5.3 umol) in 2 mL of DMSO was kept 16 hat ambient temperature, thendialyzed against water (SpectraPor 2 membrane, 12-14 kDa cutoff) toremove unconjugated materials. The solution was evaporated to dryness,and the residue was dissolved in 2 mL of THF and added slowly withstirring to 50 mL of MTBE. The precipitated conjugate was collected anddried to provide the product (140 mg). A 2.4-mg sample was dissolved in58 uL of water to give a 1 mM solution. A 20-uL aliquot was added to 100uL of 1 mM Fe(Ill) perchlorate, giving a solution showing OD42snm=0.459.Based on an extinction coefficiant of 2,300 M·1 cm·1, this indicated aDFB concentration on 1.1 mM, in good agreement with expected.

(PEG)₄₀ coupled to [DFB=Desferrioxamine B] (DFB):

PEG_(40kDa)-(DFB)₄ was prepared by reaction of PEG_(40kDa)(NH₂)₄ withp-isothiocyanatobenzyl-DFB (Perk, L. R., et al. Eur. J. Nucl. Med. Mol.I. (2010) 37:250-259; Fischer, G., et al., Molecules (2013)18:6469-6490; and van de Watering, F. C., et al. Biomed. Res. Int.(2014) 2014:203601) (macrocyclics) as follows.

4-Armed PEG_(40kDa) Coupled to DFB (PEG_(40kDa)-(DFB)4}:

A solution of 40-kDa 4-armed PEG-tetra(succinimidyl ester) (JenKemTechnologies; 100 mg, 10 umol succinimidyl ester), deferoxamine mesylate(Sigma; 10 mg, 15 umol), N,N-diisopropylethylamine (35 uL, 200 umol),and HATU (1-[Bis(dimethylamino) methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate) (7 mg, 18 umol) in 2 mLof DMF was kept 16 h at ambient temperature, then dialyzed against waterand methanol (SpectraPor 2 membrane, 12-14 kDa cutoff) to removeunconjugated materials. The solution was evaporated to dryness, and theresidue was dissolved in 2 mL of THF and added slowly with stirring to50 mL of MTBE to give the conjugate (84 mg). A 5.0 mg aliquot wasdissolved in 500 uL of water to give a solution 0.21 mM solution ofconjugate. Assay for DFB content by addition to 1 mM Fe(III) perchlorateas described above gave 0.84 μM DFB, indicating 4 DFB per conjugate.

Alternative Method

An alternative DFB reagent for conjugation, is prepared by acylation ofDFB with N₃—(CH₂)_(n)CO-HSE; the N₃—(CH₂)_(n)CO-DFB is reacted withcyclooctyne-derivatized-PEG_(40kDa)(NH₂)₄ by SPAAC.

Coupling to PET Isotopes:

Coupling to PET isotopes was performed by treatment of the PEGylated-DFBwith ⁸⁹Zr oxalate followed by purification using size-exclusionchromatography (Perk, L. R., supra; and van de Watering, F. C., supra).

PEG_(40kDa)-(DPB)₄+⁸⁹Zr-oxalate→PEG_(40kDa)-(DFB-⁸⁹Zr)₄

PEG_(40kDa)-(BzI¹²⁵I)₄ is prepared by reacting the ¹²⁵I-azide shownbelow with a cyclooctyne-derivatized-PEG_(40kDa)(NH₂)₄ (prepared fromMFCO-HSE+PEG_(40kDa)(NH₂)₄), which results in a clean high yieldstrain-promoted azide-alkyne cycloaddition (SPAAC) reaction. Preparationand radioiodination of the [¹²⁵] iodobenzoyl-PEG-azide is shown belowfor stable iodination of macromolecules using SPAAC.

Example 6 Hybrid SN38/DFB Conjugates

4-Armed PEG_(40kDa) Coupled to 1× Stable-DFB and 3× Releasable-SN-38(PEG_(40kDa)-(sDFB)₁(rSN38)₃):

A. Preparation of Hybrid SN38/DFB Conjugates.

N-((6-azidohexyloxy)carbonyl) desferrioxamine B: A solution of6-azidohexyl succinimidyl carbonate (35 mg, 120 umol) in 2 mL ofacetonitrile was added to a solution of deferoxamine mesylate (65 mg,100 umol) in 2 mL of 0.5 M NaHCO₃. After stirring for 16 h, theresulting white precipitate was collected, washed with water andacetonitrile, then dried under vacuum to yield the product (45 mg; 62%).MS: [M+H]⁺=730.46 (calc. for C₃₂H₆₀N₉O₁₀=730.44).

Azido-linker-SN38 having a cyano modulator: prepared as described in PCTPublication W02015/051307.

PEG_(40kDa)-(DBCO)₄: A solution of 40-kDa 4-armed PEG-tetraamine(PTE400-PA, NOF; 10 umol amines),dibenzocyclooctyne-N-hydroxysuccinimidyl ester (DBCO-NHS,ClickChemistryTools; 5 mg, 12 umol), and N,N-diisopropylethylamine (2uL, 12 umol) in 1 mL of acetonitrile was stirred for 1 h at ambienttemperature. The mixture was evaporated to dryness, then redissolved in1 mL of THF and precipitated by addition of 10 mL of MTBE. The resultingsolid was collected, washed with MTBE, and dried to provide the product.

PEG_(40kDa)-(sDFB)₁(rSN38)₃: A 1:3 mixture of stable-linker-DFB andreleasable-linker-SN38 was coupled to PEG_(40kDa)(DBCO)₄ to yield amixture that was predominantly PEG_(40kDa)(sDFB)₁(rSN38)₃ andPEG_(40kDa)(rSN38)₄ by HPLC analysis. These were separated bypreparative HPLC using a Phenomenex 300A 5 um Jupiter C18 column,21.2×150 mm, with a 30-60% gradient of acetonitrile in water+0.1% TFA at15 mL/min. Determination of SN38 content by UV at 360 nm (e₃₆₀=22,400M⁻¹ cm¹) and DFB content by assay with Fe(III) perchlorate as describedabove gave a 2.7:1 ratio of SN-38 to DFB.

B. Preparation of Additional Hybrid Drug/DFB Conjugates.

i. Alternate Preparation of (5HCO)₃—PEG_(40kDa)-DFB Intermediate

Step 1. (H₂N)₃—PEG_(40kDa)-NHFmoc.

A 25 mM solution of Fmoc-OSu (0.48 mL, 12 μmol) in MeCN was addeddropwise to a vigorously stirred solution of PEG_(40kDa)-(NH₂)₄ (406 mg,10.0 μmol, 5 mM final concentration) in 3.5 mL of MeCN. The reactionmixture was stirred at ambient temperature, and after 5 min, the mixtureconsisted of 44% title compound as judged by C18 HPLC (ELSD). Thereaction solution was concentrated to 1 mL by rotary evaporation. Theconcentrate was diluted to 6 mL with H₂O (0.1% TFA) then purified bypreparative C18 HPLC, two injections eluting with a linear gradient(35%-60%) of MeCN in H₂O (0.1% TFA). Fractions from the first elutingFmoc-containing peak were analyzed by C18 HPLC, and clean,product-containing fractions were combined and concentrated to dryness.After removing volatiles under high vacuum for 30 min, the residue wasdissolved in minimal THF (˜1 mL) and added dropwise to 40 mL of 0° C.MTBE in a tared 50 mL Falcon tube. The suspension was vortexed, kept onice for 15 min, centrifuged (3500× g, 1 min), and decanted. Theprecipitate was washed with MTBE (2×40 mL), isolated as above, and driedunder high vacuum to provide the title compound (96 mg, 2.2 μmol given 3TFAs, 22% yield) as a white powder. C18 HPLC, purity was determined byELSD: 99.6% (RV=9.39 mL).

Step 2. (Cyclooct-4-yn-1-yloxycarbonyl-NH)₃—PEG_(40kDa)-NHFmoc.

A 0.15 M solution of O-(cyclooct-4-yn-1-yl)-O′-succinimidyl carbonate(63 μL, 9.5 μmol) in MeCN was added dropwise to a stirred solution of(H₂N)₃—PEG_(40kDa)-NHFmoc (96 mg, 2.2 μmol, 50 mg/mL finalconcentration; 6.7 μmol NH₂) and DIPEA (2.8 μL, 16 μmol) in 1.9 mL ofMeCN. The reaction mixture was stirred at ambient temperature andmonitored by C18 HPLC. The starting material was converted to a singleproduct peak via two slower eluting intermediate peaks. After 2 h, thereaction mixture was concentrated to ˜0.3 mL by rotary evaporation. Theconcentrate was diluted with 1 mL of THF, and the solution was addeddropwise to 40 mL of ice-cold MTBE in a tared 50 mL Falcon tube. Themixture was kept on ice for 15 min then centrifuged (3500× g, 1 min) anddecanted. The wet solid was washed with ice-cold MTBE (2×40 mL),centrifuged (3500× g, 1 min) and decanted. Residual volatiles wereremoved under high vacuum for 20 min to provide the title compound (40mg, 0.93 μmol, 66% yield) as a white powder. To prevent decomposition,the solid was immediately diluted with 0.78 mL of amine-free DMF. C18HPLC, purity was determined by ELSD: 93.5% (RV=9.96 mL) and a 6.5%impurity (RV=9.78 mL).

Step 3. (Cyclooct-4-yn-1-yloxycarbonyl-NH)₃-PEG_(40kDa)-NH₂.

4-Methylpiperidine (39 μL, 5% v/v final concentration) was added to a100 mg/mL solution of(cyclooct-4-yn-1-yloxycarbonyl-NH)₃—PEG_(40kDa)-NHFmoc (0.78 mL, 78 mg,1.8 μmol) in DMF. The reaction tube was kept at ambient temperature andmonitored by C18 HPLC. After 30 min, PEG was precipitated by dropwiseaddition of the reaction solution to 40 mL of ice-cold MTBE in a tared50 mL Falcon tube. The mixture was kept on ice for 15 min thencentrifuged (3500× g, 1 min) and decanted. The wet solid was washed withMTBE (2×40 mL), centrifuged (3500× g, 1 min) and decanted. Residualvolatiles were removed under high vacuum for 15 min to provide the titlecompound (68 mg, 1.6 μmol, 89% yield) as a white powder. To preventdecomposition, the solid was immediately diluted with 0.68 mL ofamine-free DMF. C18 HPLC, purity was determined by ELSD: 87.0% (RV=9.59mL) and a 13.0% impurity (RV=9.43 mL).

Step 4.(Cyclooct-4-yn-1-yloxycarbonyl-NH)₃-PEG_(40kDa)-NHCSNH-phenyl-4-(NHCSNHDFB).

P-isothiocyanatobenzyl-desferrioxamine B (1.8 mg, 2.4 μmol;Macrocyclics) was added to a 50 mg/mL solution of(cyclooct-4-yn-1-yloxycarbonyl-NH)₃-PEG_(40kDa)-NH₂ (1.36 mL, 1.6 μmolin DMF. The reaction mixture was placed in a 37° C. water bath andmonitored by C18 HPLC. After 4 h, PEG was precipitated by dropwiseaddition of the reaction solution to 40 mL of ice-cold MTBE in a tared50 mL Falcon tube. The mixture was kept on ice for 15 min thencentrifuged (3500× g, 2 min) and decanted. The wet solid was washed withMTBE (2×40 mL), centrifuged (3500× g, 2 min) and decanted. Residualvolatiles were removed under high vacuum for 15 min to provide the titlecompound (67 mg, 1.5 mol, 94% yield) as a white solid. To preventdecomposition, the solid was immediately diluted to 2.68 mL total volumewith MeCN (2.61 mL MeCN, 25 mg/mL). Insoluble DFB-NCS was pelleted(3500× g, 2 min), and the product-containing MeCN supernatant wasremoved. C18 HPLC, purity was determined by ELSD: 80.3% (RV=9.59 mL) anda 19.7% shoulder (RV=9.43 mL).

ii. Preparation of (Drug)₃-PEG_(40kDa)-DFB

a. Drug=SN38

(SN38-L)₃-PEG_(40kDa)-NHCSNH-phenyl-4-(NHCSNH-DFB).

Stable azido-SN38 (4.0 mg 5.2 mol, 4 mM final concentration; Santi etal., J. Med. Chem. 57: 2303-14 (2014)) was added to a 25 mg/mL solutionof(cyclooct-4-yn-1-yloxycarbonyl-NH)₃—PEG_(40kDa)-NHCSNH-phenyl-4-(NHCSNHDFB)(1.3 mL, 0.75 μmol PEG, 2.3 mol cyclooctyne, 1.8 mM cyclooctyne finalconcentration) in MeCN. The reaction was placed in a 37° C. water bathand monitored by C18 HPLC. After 44 h, the reaction solution wasdialyzed against MeOH (12-14 k MWCO). The dialysate was concentrated todryness, and residual volatiles were removed under high vacuum toprovide the title compound (24 mg, 0.52 μmol, 69% yield by mass) aswhite film that contained 1.4 mol of SN38 as determined by A₃₈₃ and 0.50μmol of DFB as determined by A₄₉₀ of Fe³⁺-DFB. The SN38:DFB ratio wasfound to be 2.8:1 using SN38 ε₃₈₃=29,100 M⁻¹ cm⁻¹ and Fe³⁺-DFBε₄₉₀=3,000 M⁻¹ cm⁻¹. C18 HPLC, purity was determined by ELSD: 83.0%(RV=9.67 mL) and a 14.6% shoulder (RV=9.52 mL).

b. Drug=Rucaparib a PARP Inhibitor

(Rucaparib-L)₃-PEG_(40kDa)-NHCSNH-phenyl-4-(NHCSNH-DFB).

A 10 mM solution of stable azido-rucaparib (0.11 mL, 1.1 mol, 1.8 mMfinal concentration; prepared by reacting rucaparib with 6-azidohexylsuccinimidyl carbonate according to the procedures of Santi et al.,Proc. Natl. Acad. Sci. 109: 6211-16 (2012)) was added to a 25 mg/mLsolution of(cyclooct-4-yn-1-yloxycarbonyl-NH)₃—PEG_(40kDa)-NHCSNH-phenyl-4-(NHCSNHDFB)(0.50 mL, 0.29 umol PEG, 0.86 umol cyclooctyne, 1.4 mM cyclooctyne finalconcentration) in MeCN. The reaction was placed in a 37° C. water bathand monitored by C18 HPLC. After 68 h, the reaction solution contained a35:65 mixture of unmodified:PEGylated drug-linker. A series of theindividual species of (drug)n-PEG-DFB was not observed. The reactionsolution was concentrated by SpeedVac to 0.1 mL, diluted to 1.0 mL withH₂O, and loaded onto a PD-Midi column. Elution with H₂O yielded a cloudyfraction of excluded material that contained both unmodified andPEGylated drug-linker. The mixture was then dialyzed against MeOH (12-14k MWCO). The dialysate was concentrated to dryness, and residualvolatiles were removed under high vacuum to provide the title compound(8.7 mg, 0.19 umol, 66% yield) as white film that contained 0.51 μmol ofrucaparib as determined by A₃₅₅ and 0.19 umol of DFB as determined byA₄₉₀ of Fe³⁺-DFB. The rucaparib:DFB ratio was found to be 2.7:1 usingrucaparib ε₃₅₅=13,260 M⁻¹ cm⁻¹ (125SF68) and Fe³⁺-DFB ε₄₉₀=3,000 M⁻¹cm¹. C18 HPLC, purity was determined by ELSD: 78.5% (RV=9.41 mL) and a21.5% shoulder (RV=9.27 mL).

c. Drug=VX-970—an ATR Kinase Inhibitor

(VX970-L)₃-PEG_(40kDa)-NHCSNH-phenyl-4-(NHCSNH-DFB).

As described above for rucaparib, stable azido-VX970 (0.11 mL, 1.1 umol,1.8 mM final concentration; prepared by reacting VX970 with 6-azidohexylsuccinimidyl carbonate according to the procedures of Santi et al.,Proc. Natl. Acad. Sci. 109: 6211-16 (2012)) was treated with a 25 mg/mLsolution of(cyclooct-4-yn-1-yloxycarbonyl-NH)₃—PEG_(40kDa)-NHCSNH-phenyl-4-(NHCSNHDFB)(0.50 mL, 0.29 umol PEG, 0.86 umol cyclooctyne, 1.4 mM cyclooctyne finalconcentration) in MeCN to provide the title compound (10 mg, 0.22 umol,76% yield by mass) as white film that contained 0.55 umol of VX970 asdetermined by A₃₈₃ and 0.24 μmol of DFB as determined by A₄₉₀ ofFe³⁺-DFB. The VX970:DFB ratio was found to be 2.3:1 using VX970ε₃₈₃=17,200 M⁻¹ cm¹ (127BH52) and Fe³⁺-DFB ε₄₉₀=3,000 M⁻¹ cm⁻¹. C18HPLC, purity was determined by ELSD: 59.2% (RV=9.98 mL) and a 38.4%shoulder (RV=9.73 mL).

d. Drug=BMN673-a PARP Inhibitor

(BMN673-L)₃-PEG_(40kDa)-NHCSNH-phenyl-4-(NHCSNH-DFB).

As described above for rucaparib, stable azido-BMN673 (0.11 mL, 1.1μmol, 1.8 mM final concentration; prepared by reacting BMN673 with6-azidohexyl succinimidyl carbonate according to the procedures of Santiet al., Proc. Natl. Acad. Sci. 109: 6211-16 (2012)) was treated with a25 mg/mL solution of(cyclooct-4-yn-1-yloxycarbonyl-NH)₃—PEG_(40kDa)-NHCSNH-phenyl-4-(NHCSNHDFB)(0.50 mL, 0.29 mmol PEG, 0.86 μmol cyclooctyne, 1.4 mM cyclooctyne finalconcentration) in MeCN to provide the title compound (12 mg, 0.26 μmol,91% yield by mass) as white film that contained 0.65 μmol of BMN673 asdetermined by A₃₁₀ and 0.20 μmol of DFB as determined by A₄₉₀ ofFe³⁺-DFB. The BMN673:DFB ratio was found to be 3.3:1 using BMN673ε₃₁₀=9872 M⁻¹ cm⁻¹ (125SF39) and Fe³⁺-DFB ε₄₉₀=3,000 M⁻¹ cm⁻¹. C18 HPLC,purity was determined by ELSD: 69.7% (RV=9.47 mL) and a 30.3% shoulder(RV=9.32 mL).

C. Coupling to PET Isotopes

The hybrid SN38/DFB and alternative hybrid drug/DFB conjugates arecoupled to ⁸⁹Zr by the methods set forth in Example 5.

Example 7 Use of PET to Detect EPR in Animal Studies

Mice bearing HT-29 human xenografts and normal mice are treated withconjugates PEG-PET isotopes which are similar in size and shape to thedrug conjugates of Examples 1-4. PET-imaging to measure accumulation oflabeling intensity of the tumor at t=0, 12, 24, 48 and 96 hr isconducted in comparison with results of a similar experiment usingPEG_(40kDa)-fluorescein in tumor-bearing mice (Singh, Y., supra). Seraare counted at these time points to determine the t_(1/2) of eliminationof the PEG-isotope (the elimination t_(1/2) of PEG_(40kDa) in mice isusually 24 hr), as well as total body radioactivity measurements.

HT-29 Tumor bearing mice and normal control mice are treated with ˜200uCi/mouse, and PET-imaging is performed at varying times to determinethe amount and rates of accumulation. A signal is observable at ˜1uCi/cc so the tumor is easily visualized as long as the backgroundtissue does not accumulate the tracer. In the same experiment, the lossof isotope is followed as the reagent is cleared from the body. Rates ofa) tumor accumulation of the PEG-isotope (quantitative PET imaging), b)vascular elimination (serum radioactivity), c) systemic elimination(whole body radioactivity) and d) tumor elimination (quantitative PETimaging) are thus determined.

At a time when tumor accumulation is complete, tumor-bearing mice aretreated with varying amounts of the PEG_(40kDa)-isotope to determine themaximal amount of nanoparticle that can accumulate.

Thus, in this example, PET scanning is used to simulate the behavior ofan agent coupled to the same or similar carrier to evaluate theparameters appropriate for the drug administration protocol.

Example 8 PET Imaging/Biodistribution of PEG_(40kDa)-DFB⁸⁹Zr

Mice bearing xenografts (n=5) were injected with ˜300 μCi (8.4 nmol) ofPEG_(40kDa)-DFB-⁸⁹Zr and microPET/CT images were obtained at 24 h (n=2)and 48 h (n=2). The % ID/g uptake (uptake of PEG_(40kDa)-DFB-⁸⁹Zr) intumors was 15 and 20% at 24- and 48 h, respectively, while organs otherthan liver had ≤3% uptake. MicroPET/CT studies showed high accumulationof ⁸⁹Zr-DFB-PEG₄₀ in MX-1 tumors as early as 24h while accumulation inhealthy tissue was nearly background. The imaging data corroborated theincreased accumulation in tumor from 24 to 48h. However, there washeterogeneous uptake in the tumor, possibly suggesting necrosis of thisrapidly growing tumor.

The experiment was repeated the slower growing HT-29 tumor. Given thelower tumor to blood ratios and limited clearance at early time pointsin MX-1 tumors (1.1±0.2 [24 h]-1.2±0.1 [48 h]) the uptake in the HT-29tumors was studied at 72 h and 120 h. Mice (n=8) were injected with 160μCi (8.4 nmol) of ⁸⁹Zr-DEB-PEG₄₀ and microPET/CT images were obtained at72- and 120h. Mice were euthanized at 72- and 120h for ex-vivobiodistribution studies. HT-29 tumors were clearly visualized on themicroPET/CT at 72h and 120h (FIG. 6A), and biodistribution studiesrevealed high uptake of 20.6±2.4 and 14.4±4.5% ID/g at 72 and 120h (FIG.6B) with tumor/blood 2.8±0.4 and 5.1±1.3 at 72 and 120h, respectively(FIG. 6C). FIG. 6D is an MIP image of PEG-SN-38)₃-DFB⁸⁹Zr in a singleflank tumor-bearing mouse. FIG. 6E shows biodistribution ofPEG-(SN-38)₃-DFB-⁸⁹Zr (black) vs PEG-DFB-⁸⁹Zr (grey) at 72h.

In an additional study, the PEG_(40kDa)-(DFB-⁸⁹Zr)₄ of Example 5 wasinjected into mice bearing HT29 tumors. Five mice were used in the studyand each was injected with 250-290 μCi of the conjugate in 100 μlsaline. Two of the mice were imaged at one hour post injection. After 24hours, two mice, (one that had been imaged at one hour and an additionalmouse) were imaged and then sacrificed to perform distribution studies.At 48 hours, two mice were imaged (one of the mice that was imaged atone hour and one additional mouse) and these were also sacrificed alongwith the remaining mouse and a distribution study performed.

The results of these studies are shown in FIGS. 7A-7C. Shown in FIG. 7A,the label was present in the tumor at all of the times measured. Asshown in FIG. 7B, the % of the injected dose (ID) per gram of individualorgans was significant in most organs, although bone, spleen and tumorhad the highest levels. As shown in FIG. 7C when computed as thepercentage of the injected dose per organ, rather than as per gram oforgan, accumulation in the tumor was dramatically higher, especially at48 hours, as compared to other organs. Only liver showed a significantaccumulation which dropped over the time period of 24-48 hours. Thus,the imaging agent confirms that the conjugate is selectively accumulatedin the tumor as compared to other organs.

Example 9 Additional Distribution Studies

The experiments of Example 8 were repeated using 4-branchedPEG_(40kDa)-DFB-⁸⁹Zr (Example 5), 4-armed PEG_(40kDa)-(DFB-⁸⁹Zr)₄(Example 5), and 4-armed PEG_(40kDa)-(DFB-⁸⁹Zr)₁(SN38)₃ (Example 6) inboth MX-1 and HT-29 xenografts. PET imaging was used to measureaccumulation of ⁸⁹Zr in tumor, heart, liver, and kidney at 1, 24, 48,72, 96, and 216 h post-dose. The resulting data (expressed asdecay-corrected percent of the total dose) were analyzed using amembrane-limited tissue distribution model according to the methods ofLi et al., Intl. J. Nanomedicine (2012) 7: 1345-56. A compartment forthe remaining tissues was included in order to match measured bloodlevels in the absence of more specific tissue analyses. Blood data werefit using a total clearance equal to the sum of the diffusioncoefficients from blood into the organs (k, Table 2) and the eliminationrate constant calculated from a plasma half-life of 20 hours.

Within experimental error, all three compounds showed the same tissuedistribution in a specific tumor xenograft. FIG. 8 shows thedistribution of ⁸⁹Zr in HT-29 xenografts, and FIG. 9 shows thedistribution of ⁸⁹Zr in MX-1 xenografts. Model parameters are given inTable 2, where R=tissue-blood partition coefficient, k=diffusioncoefficient, V=tissue volume, and VVF=the vascular fraction of thetissue.

TABLE 2 Parameters for Membrane-Limited Tissue Distribution Model R k(h⁻¹) V (mL) VVF k/RV HT-29 Circulation 2.8 1 Heart 0.7 0.0015 0.15 0.230.0143 Kidney 0.6 0.0017 0.5 0.08 0.00567 Liver 1.5 0.013 1.65 0.150.00523 Tumor 5 0.0095 1 0.04 0.0019 Body 1 0.03 30 0.1 0.001 MX-1Circulation 2.8 1 Heart 0.7 0.0015 0.15 0.23 0.0143 Kidney 0.5 0.00150.5 0.09 0.006 Liver 1.2 0.012 1.65 0.134 0.00606 Tumor 5 0.0062 0.450.075 0.00276 Body 1 0.03 30 0.1 0.001

In both xenograft models, the ⁸⁹Zr-conjugates were observed toaccumulate selectively in the tumor tissue and be retained for muchlonger times than in other tissues.

Example 10 Correlation of Biodistribution of Imaging Agent and ActiveAgent

In this example, the pharmacokinetics/biodistribution of the imagingagent PEG_(40kDa)-DFB⁸⁹Zr is compared with that of PEG-SN-38.

SN-38 is the active metabolite of irinotecan (CPT-11) a widely usedanticancer agent. (PEGSN-38) is a conjugate of 4 arm PEG_(40kDa) with 4equivalents of SN-38, giving PEG_(40kDa)(SN-38)₄ (Santi D V, et al., J.of Med. Chem. (2014) 57(6):2303-2314). (PEGSN-38 is in dose escalationin Phase 1 trials and shows a long t_(1/2,β) of 6 days.)

Xenograft mice are prepared by implantation of 10⁶ to 10⁷ HT29 cellsinto the NSG mouse flank, and maintained until the tumors are ˜200 mm³.Time vs activity curves from microPET/CT images, blood, tumor and mainorgans are used to determine the accumulation/elimination rates ofPEG_(40kDa)-DFB-⁸⁹Zr in the tumor, the elimination rate from the bloodand body, and the temporal activity distribution in the remainder of themouse. Increasing concentrations of PEG_(40kDa)-DFB-⁸⁹Zr increase therate of accumulation, with no effect on the first-order elimination fromtumors.

Varying doses of the unlabeled PEG˜(SN-38)₄ conjugate are injected intoanimals. From preclinical toxicology studies of PEG˜(SN-38), the dose toprovide 50% tumor growth inhibition (TGI) in the HT-29 tumor/nude ratewas 150 mg/kg. From allometric scaling, 50% TGI in the mouse should be280 mg/kg. A target dose for measurable growth inhibition (e.g. ˜50%TGI) is verified.

A mixture of PEG˜(SN-38)₄ and PEG-(DFB-⁸⁹Zr) is prepared that suitablefor both a) achieving the therapeutic target dose, and b) monitoringtumor uptake/elimination kinetics of PEG-(DFB-⁸⁹Zr) measured by PET over10 days, as described above. Tissues are removed to quantifybiodistribution, blood sampling. Total SN-38 content of tumors ismeasured by HPLC of NaOH-digested tumor and blood samples at varioustimes (Santi, et al. (supra)). The PEG˜(SN-38)₄/PEG-(DFB-⁸⁹Zr) ratio isdetermined at various time points to verify either an identity ofdrug/isotope of the ratio vs time or other relationship of tumor uptakeof two components.

The % ID/g tumor of PEG-(DFB-⁸⁹Zr) that corresponds to a therapeuticdose of PEG˜(SN-38)₄ is established. High-uptake tumors are identifiedthat accumulate sufficient PEG˜(SN-38)₄ to achieve a therapeutic dose.

Thus, the subjects who will benefit from an EPR effect of a conjugatedSN-38 are identified by an initial administration of the imaging agentof the invention.

Example 11 Efficacy of PLX038A

The SN38 conjugate designated PLX038A in Example 1 and abbreviated hereas PEG-SN38 is used in this Example.

Four groups of mice having 5 mice in each group bearing MX-1 tumorxenographs were injected with vehicle or with a single dose of eithervehicle, 137 mole/kg irinotecan (0.137/g or ˜4 mole per mouse) or with120 mole/kg PEG-SN38 qdx x 1d (single dose). Tumor volume was measuredas a function of time. At 42 days, the group that received vehicle wastreated with 120 μmole/kg of PEG-SN28. The results are shown in FIG. 10.

As shown, MX-1 tumor growth in the mice injected with vehicle continuedapace, reaching 1200 mm³ after 4 weeks, for the initial 42 days untilthe PEG-SN38 was injected whereupon the tumor volume declineddramatically. Dosage at time 0 with PEG-SN38 immediately eliminated thetumor. Irinotecan, while having some effect, was only somewhat betterthan vehicle after 4 weeks these tumors reached 600 mm³.

Further, for mice with untreated tumors that showed tumor growth even aslarge as 1.7 cm³, a single MTD dose of PEGSN38 shrank these tumors.

These results demonstrate that PEG-SN38 is highly effective for treatingsolid tumors and that the findings with the imaging agent in Example 8are consistent with this result.

Example 12 Synergistic Effect of PLX038A and PARP Inhibitor Talazoparib(Designated BMN673 or TLZ)

Preparation of murine MX-1 xenografts: The MX-1 cell line was obtainedfrom Charles River Labs (Frederick, Md.). Ovejera A A et al. Ann ClinLab Sci (1978) 8:50-6. Cells were cultured in RPMI-1640, 10% FBS and 1%2 mM L-glutamine at 37° C. in 95% air/5% CO₂ atmosphere.

Female NCr nude mice (N CrTac:NCr-Foxn1^(mi); ˜6-7 weeks old) fromTaconic Bioscience (Cambridge City, Ind.) were housed at the UCSFPreclinical Therapeutics Core vivarium (San Francisco, Calif.). Allanimal studies were carried out in accordance with UCSF InstitutionalAnimal Care and Use Committee. Tumor xenografts were established bysubcutaneous injection with MX-1 tumor cells (2×10⁶ cells in 100 μI ofserum free medium mixed 1:1 with Matrigel) into the right flank offemale NCr nude mice. When tumor xenografts reached 1000-1500 mm³ indonor mice, they were resected, cut into even-size fragments(˜2.5×2.5×2.5 mm in size), embedded in Matrigel and re-implanted viasubcutaneous trocar implantation in receiver mice. Morton C L, HoughtonP J. Nat Protoc. (2007) 2:247-50.

Dosing and Tumor Volume Measurements:

Solutions of PLX038A (1.02 mM SN38; 0.26 mM PLX038A conjugate) wereprepared in pH 5 isotonic acetate and sterile filtered (0.2 um) beforeuse. Solutions of BMN673 (52 μM) were prepared in 10%dimethylacetamide/5% Solutol HS15/85% 1×PBS and were sterile filtered(0.2 um) before use.

Groups (N=4-5/group) were dosed when the group average reached 100-200mm³ in size. Mice received vehicle, a single dose of PLX038A (14.7 mL/kgi.p., 15 μmol/kg), daily doses of BMN673 (7.72 mL/kg p.o., 0.4 umol/kg),or a combination of PLX038A and BMN673 at the same doses. For groupsreceiving the combination, daily BMN673 dosing began on the same day(FIG. 11A) or after a 4-day delay (FIG. 11B) after dosing PLX038A. Tumorvolumes (caliper measurement: 0.5×(length×width)) and body weights weremeasured twice weekly. When vehicle control tumors reached 3000 mm³ insize, mice were treated with the combination of a single dose of PLX038A(15 umol/kg) and daily BMN673 (0.4 umol/kg) combination with no delaybetween dosing (FIG. 11A).

As shown in FIGS. 11A and 11B, administration of PLX038A to mice bearingMX-1 tumors at 15 umol/kg in combination with daily doses of Talazoparibat 0.4 umol/kg provides a synergistic effect as compared to either ofthese drugs alone. This was true whether daily dosage with TLZ began atthe same time as PLX038A or 4 days later. A single combinationadministered to control immediately reduced tumor volume (FIG. 11A).

As shown in FIG. 11C, event-free survival was enhanced synergisticallywith the combination vs PLX038A and TLZ individually.

1. A method to ameliorate the toxicity to normal tissue in a subjectresulting from administering to said subject a first and secondchemotherapeutic agent in a protocol for combination therapy against asolid tumor employing said first and second agent, which methodcomprises: administering the first agent as an agent-releasing conjugateto a flexible carrier wherein the carrier is a nanoparticle ormacromolecule each with a hydrodynamic radius of 5-50 nm which conjugateexhibits enhanced permeability and retention (EPR) in solid tumors so asto concentrate said conjugate in the tumor and wherein the rate ofrelease from the tumor of the conjugate and first agent released fromthe conjugate is substantially slower than the rate of clearance of theconjugate and released agent from the systemic circulation of thesubject; allowing a time period for clearance of the conjugate andreleased agent from the systemic circulation of the subject; and aftersaid time period, administering said second agent to the subject.
 2. Themethod of claim 1, wherein the second agent is administered in freeform, or wherein the second agent is administered as an agent-releasingconjugate to a carrier, wherein the carrier is a nanoparticle ormacromolecule each with a hydrodynamic radius of 5-50 nm.
 3. The methodof claim 1, which further includes administering a third agent withnon-overlapping toxicity with the second agent.
 4. The method of claim1, which further includes allowing a time period for clearance of thesecond agent; and after said time period, again administering saidconjugated first agent to the subject.
 5. The method of claim 1, whereinthe characteristics associated with the concentration of the conjugatein the solid tumor are measured by administering a label non-releasablycoupled to the same carrier as the first agent and tracking the label invivo in said subject.
 6. The method of claim 5, wherein the label is anisotope detectable by positron emission tomography (PET) scanning. 7.The method of claim 1, wherein the conjugate releases said first agentby beta elimination or by hydrolysis of esters, carbonates, orcarbamates, or by proteolysis of amides or by reduction of aromaticnitro groups by nitroreductase.
 8. The method of claim 1, wherein thecarrier comprises a polyethylene glycol of molecular weight 10 kD-60 kD.9. The method of any of claims 1-8, wherein the first agent is atopoisomerase inhibitor, an anthracycline, a taxane, an epothilone, atyrosine kinase inhibitor, an inhibitor of homologous recombinationrepair, a biologic, an anti-steroid, or a nucleoside.
 10. The method ofclaim 9, wherein the first agent is a topoisomerase inhibitor.
 11. Themethod of any of claims 1-8, wherein the second agent is an inhibitor ofhomologous recombination repair, an agent synergistic to or additive toa PARP inhibitor, or an mTOR inhibitor, trabectedin, cis-platinum,oxaliplatin, fluorouracil, temozolomide or vincristine.
 12. A method tominimize the toxic effects on normal tissue of a subject of a first andsecond chemotherapeutic agent used in combination to treat a solid tumorin said subject which method comprises administering said second agentsimultaneously with said first agent, said first agent being in the formof a conjugate to a flexible carrier, wherein said conjugate exhibitsenhanced permeability and retention (EPR) and effects concentration ofsaid conjugate in said tumor, wherein the carrier is a nanoparticle ormacromolecule with a hydrodynamic radius of 5-50 nm.
 13. The method ofclaim 12, wherein the second agent is conjugated or unconjugated. 14.The method of claim 12, wherein the second agent is conjugated to acarrier with the same structure as the carrier for the first agent. 15.The method of claim 12, wherein the characteristics associated with theconcentration of the conjugate(s) in the solid tumor are measured byadministering a label non-releasably coupled to the same carrier as thatfor at least the first agent and tracking the label in vivo in saidsubject.
 16. The method of claim 15, wherein the label is an isotopedetectable by positron emission tomography (PET) scanning.
 17. Themethod of claim 14, wherein the conjugate(s) release said agents by betaelimination or by hydrolysis of esters, carbonates, or carbamates, or byproteolysis of amides or by reduction of aromatic nitro groups bynitroreductase.
 18. The method of claim 12, wherein the macromolecularcarrier(s) comprise(s) polyethylene glycol of molecular weight of 10kD-60 kD.
 19. The method of any of claims 12-18, wherein the first agentis a topoisomerase inhibitor, an anthracycline, a taxane, an epothilone,a tyrosine kinase inhibitor, an inhibitor of homologous recombinationrepair, a biologic, an anti-steroid, or a nucleoside.
 20. The method ofclaim 19, wherein the first agent is a topoisomerase inhibitor.
 21. Themethod of any of claims 12-18, wherein the second agent is an inhibitorof homologous recombination repair, an agent synergistic to or additiveto a PARP inhibitor, or an mTOR inhibitor, trabectedin, cis-platinum,oxaliplatin, fluorouracil, temozolomide or vincristine.
 22. An imagingagent of the formula (1)

wherein PEG represents a polyethylene glycol comprising a plurality of2-6 arms of 40-60 kD; chelator represents a desferrioxamine or aplur-hydroxypyridinone multidentate; I is a radioisotope suitable forpositron emission tomography (PET);

is a covalent connector; ˜ indicates sequestration of I in the chelator;and n is an integer of 1 up to the number of arms of said PEG.
 23. Theimaging agent of claim 22 wherein I is ⁸⁹Zr, ⁹⁴Tc, ¹⁰¹In, ⁸¹Rb, ⁶⁶Ga,⁶⁴Cu, ⁶²Zn, ⁶¹Cu or ⁵²Fe; and/or wherein PEG is a four armedpolyethylene glycol of approximately 40 kD, and n is 1-4; and/or whereinthe chelator is desferrioxamine-B; and/or wherein

is a direct bond linkage.
 24. A method to monitor accumulation of theimaging agent of claim 22 or 23 in a tumor which method comprisesadministering said imaging agent and detecting the location of saidimaging agent by PET.
 25. A method to assess the pharmacokinetics of theconjugate of a drug and its accumulation in tumor which method comprisesmatching the size and shape of the conjugate of said drug to the sizeand shape of the imaging agent of claim 22 or 23, administering saidimaging agent to a subject bearing a tumor and monitoring theaccumulation of said agent in the tumor by PET.
 26. A kit that includesthe imaging agent of claim 22 or 23 and a drug conjugate.
 27. A methodto identify a subject having an undesirable tissue mass likely tobenefit from treatment with a drug modified to exhibit the EPR effect,which comprises administering the imaging agent of claim 22 or 23 to acandidate subject; and monitoring the distribution of the imaging agentin the subject, whereby a subject that accumulates said imaging agent insaid undesirable tissue mass is identified as a subject that willbenefit from such treatment.
 28. The method of claim 27 which furtherincludes determining the presence or absence of a mutation in a genethat encodes a protein that participates in effecting DNA repair,wherein the presence of said mutation in the subject identifies thesubject as having said tumor.
 29. The method of claim 28 wherein thegene is BRCA1, BRCA2, ATM or ATR.
 30. A hybrid conjugate for treatmentand imaging of solid tumors which conjugate comprises a flexible carrierwherein the carrier is a nanoparticle or macromolecule each with ahydrodynamic radius of 5-50 nm which conjugate exhibits enhancedpermeability and retention (EPR) in solid tumors so as to concentratesaid conjugate in the tumor and wherein said carrier is releaseablycoupled to a therapeutic agent and also coupled to an imaging agent. 31.The hybrid conjugate of claim 30 which is of formula (2)

wherein PEG represents a polyethylene glycol comprising a plurality of2-6 arms of 40-60 kD; chelator represents a desferrioxamine or aplur-hydroxypyridinone multidentate; I is a radioisotope suitable forpositron emission tomography (PET);

is a covalent connector; ˜ indicates sequestration of I in the chelator;L is a linker; D is a therapeutic agent; n is an integer of 1 up to thenumber of arms of said PEG minus x; and x is an integer of up to thenumber of arms of said PEG minus n.
 32. The imaging agent of claim 31wherein I is ⁹⁸Zr, ⁹⁴Tc, ¹⁰¹In, ⁸¹Rb, ⁶⁶Ga, ⁶⁴Cu, ⁶²Zn, ⁶¹Cu or ⁵²Fe;and/or wherein PEG is a four armed polyethylene glycol of approximately40 kD, and n is 1-4; and/or wherein the chelator is desferrioxamine-B;and/or wherein

is a direct bond linkage; and/or D is SN38, BMN673, VX-970 or rucaparib.33. A method to correlate imaging and treatment of a solid tumor whichmethod comprises administering to a solid tumor-bearing subject thehybrid conjugate of any of claims 30-32 and monitoring the accumulationof said conjugate in the tumor and monitoring the volume of said tumor.